The sole tryptophan of amicyanin enhances its thermal stability but does not influence the electronic properties of the type 1 copper site

Abstract

The cupredoxin amicyanin possesses a single tryptophan residue, Trp45. Its fluorescence is quenched when copper is bound even though it separated by 10.1 Å. Mutation of Trp45 to Ala, Phe, Leu and Lys resulted in undetectable protein expression. A W45Y amicyanin variant was isolated. The W45Y mutation did not alter the spectroscopic properties or intrinsic redox potential of amicyanin, but increased the pKa value for the pH-dependent redox potential by 0.5 units. This is due to a hydrogen-bond involving the His95 copper ligand which is present in reduced W45Y amicyanin but not in native amicyanin. The W45Y mutation significantly decreased the thermal stability of amicyanin, as determined by changes in the visible absorbance of oxidized amicyanin and in the circular dichroism spectra for oxidized, reduced and apo forms of amicyanin. Comparison of the crystal structures suggests that the decreased stability of W45Y amicyanin may be attributed to the loss of a strong interior hydrogen bond between Trp45 and Tyr90 in native amicyanin which links two of the β-sheets that comprise the overall structure of amicyanin. Thus, Trp45 is critical for stabilizing the structure of amicyanin but it does not influence the electronic properties of the copper which quenches its fluorescence.

Intrinsic Tryptophan Fluorescence in the Detection and Analysis of Proteins: A Focus on Förster Resonance Energy Transfer Techniques

“…Fluorescence quenching can also arise via a resonance energy transfer mechanism, with a suitable acceptor molecule or metal ion. Cupredoxins, which possess a Trp residue located within 1 nm of the copper ion, e.g., azurin [ 51 ], stellacyanin [ 52 ] and amicyanin [ 53 ], exhibit the phenomenon of Trp fluorescence quenching upon metal ion binding via either Förster resonance energy transfer or an electron transfer mechanism [ 54 , 55 ].…”

Relaxation of structural constraints during Amicyanin unfolding

“…Results to date indicate that in all cases the copper center stabilizes residues constituting the cupredoxin fold, and that this stability persists for most (but not all) of the residues as the protein unfolds. Of relevance here is that copper-induced protein stabilization has been documented experimentally by Dow et al [43].…”

“…This finding was unexpected due to similarities in crystal structures. Davidson et al [24,43,47] argue that the removal of copper may modify the charge distribution on the surface of the protein and that this in turn may affect its ability to bind to other proteins as well as to self-associate. Regarding the thermal stability of the protein, studies [23,30,47] show that this property also depends on the presence or absence of copper and its ligand geometry, but not the metal redox state.…”

“…For example, using Chimera, we examined the "match" between our protein [PDB# 1AAC] and reduced W45Y amicyanin [PDB# 4P5S] studied by Dow et al [43]. The graphics reveal that the residue in question, Trp45, and the mutation, W45Y, overlap almost perfectly.…”

“…Of special interest is that these mutations impact the hydrogen bonding network as the protein unfolds [43].…”